This proposal is concerned with an examination of the energetics and the cellular organization of H ion transport by an in vitro urinary epithelium, the bladder of the fresh water turtle. This preparation provides the opportunity to examine both the metabolic function of the epithelium and its capacity to transport H ion. Previous studies have demonstrated that H ion transport in this tissue is coupled to the rate of glucose oxidation by the pentose phosphate shunt. Therefore it is proposed that H ion transport is dependent in some manner on NADPH generated by this pathway and in turn the utilization of NADPH controls the rate of pentose shunt metabolism. To evaluate this proposal the following will be examined: (1) the relationship between the rate of H ion transport, pentose metabolism and the NADP/NADPH ratio; (2) the effect of depleting NADPH with oxidizing agents on H ion transport and pathway of glucose oxidation and (3) the capacity of partially purified apical plasma membranes to oxidize NADPH or hydrolyze ATP. The mucosal surface cells of the turtle bladder consist of mitochondria-rich cells and granular cells. The mitochondria-rich cell may be the primary H ion transport cells because they have increased carbonic anhydrase and G6PD activity when compared to granular cells. To demonstrate this cell specialization we plan to separate the cells into relatively purified subpopulations and then examine the effect of specific transport inhibitors on their metabolic rate, their ability to generate pH gradients and modulate their Na ion content and lastly the distribution of bindings sites on these cells for inhibitors of H ion transport that form covalent bonds with the membrane. Lastly because of heterogeneity among bladders and the paucity of material available after cell fractionation we plan to develop a turtle bladder-derived cell line in culture that has the capacity to transport H ion.